This proposal is aimed at achieving a better understanding of the neural components of the stress system, both in terms of the key molecules which control stress responses and in terms of the critical circuits involved in the initiation and the termination of these responses. While a great deal is known about the peripheral elements of the limbic-hypothalamo-pituitary- adrenal (LHPA) axis, somewhat less is known about the way in which the brain controls the functioning of this axis. Of particular interest are the glucocorticoid negative feedback mechanisms which are essential in limiting and terminating the stress response. These feedback mechanisms depend, to a large extent, on limbic corticosteroid receptors which appear to exert their effects both at the cellular level via genomic mechanisms, and at the multicellular level by influencing neural circuits which mediate negative feedback. In this project we shall study at the molecular level the two known types of corticosteroid receptors involved in these processes - the glucocorticoid receptor (GR), and the mineralocorticoid receptor (MR), in an effort to understand the functional significance of their dual control over the axis. By using site directed mutagenesis, we shall investigate the molecular basis for the selectivity of these receptors towards their steroid ligands, and the basis of their coupling to a heat shock protein (hsp-90) complex which is required for their ligand binding and consequent translocation to the nucleus. We shall compare the way in which these two receptors, which are transacting factors, interact with recognition elements on target genes, in order to ascertain whether they share identical or somewhat divergent targets. Finally, we shall study whether multiple forms of MR mRNA are differentially translated or whether they are differentially expressed in specific neurons. To date, our analysis of the neuronal circuit which mediates negative feedback indicates a bisynaptic pathway originating at the level of the hippocampus - an area rich in GR and MR- and terminating in the paraventricular nucleus (PVN), which harbors the releasing factors, by way of the bed nucleus of the stria terminalis. The biochemical anatomy of this circuit will be investigated to determine the nature of the neurotransmitters involved in stress termination. In order to begin to delineate the circuits involved in initiation of the stress response, we shall employ molecular methods for detecting rapid activation of the relevant neuronal elements, including the visualization of immediate early genes (c-Fos, c-Jun), and, by using intronic in situ hybridization, the detection of primary transcripts of stress-related genes which reflect recent transcription. These tools, in combination with lesions, transections and classical endocrine measures, will be applied towards identifying important inputs to the PVN which are essential for stress responsiveness. Finally, in a more integrative vein, we shall describe with molecular biological, biochemical, anatomical and neuroendocrine tools the altered regulation of the stress axis observed in aged rats. This naturalistic model should help us to better understand the interplay between the molecular and neuronal elements of the axis. It also has direct relevance to the investigation of the axis in aging and depression, which is undertaken in Project IV.